In article <2c8tki$hfd at news.bu.edu>, reichert at bu.bu.edu (Tanya Reichert) writes:
> For the past month, I've been trying work out a technique for detecting
> copy number of a certain gene from genomic DNA of varied animal species
> via PCR. The idea is to take flanking exon sequences that are very well
> conserved across species, construct degenerate primers from them, and
> amplify the intron that they delimit. Assuming that the intron size varies
> from gene to gene within a species, the number of PCR products should
> reflect the copy number of the gene.
>> OK... nice idea, here's the problem. In species I know contains three
> copies of my gene, I can't get all three bands (representing the three
> genes) to show up at the same temperature. I get the two smaller ones
> at lower temperatures and, sometimes, I get the largest one at relatively
> higher temps.
>> I've tried doing MgCl2 and DNA concentration titrations. I've also tried
> cutting the DNA with a 7-cutter (to try and make the sites more accessible
> to the primers) and ran PCR on unextracted and extracted DNA from those
> reactions. All to no avail.
>> Any ideas? Could it be something funky with the intron? Am I wasting
> my time?
>> Thanks for your help.
>> Tanya
> e-mail: reichert at bio.bu.edu
Hi Tanya!
In the past I was using this method trying to detect the number of HMGR encoding
isogenes in potato. What I did is that I used a set of primers (made originally
for tomato) which were derived from a region highly conserved between HMGRs and
thus likely to anneal to multiple HMGR genes and they span two introns making
it more likely to see differences between isogenes.
Based on previous results and what we did ourselves, we knew that potato has
at least four HMGR encoding isogenes (but likely there is going to be more).
When analyzing PCR-reactions, we were able to see six bands of different size.
But in the same reaction it was possible to get at the maximum four different
bands, usually even less. We also noticed that the temperature and MgCl2
concentrations were the most important things affecting to the number of
yielding bands.
We used annealing temperatures from 32 to 58 C, when running those reactions.
Depending of the temperature we were able to detect few faint bands (usually
lower temperatures) or at higher temperatures just one or two really bright
band(s). Because potato is a tetraploidy plant, it really creates some other
problems that you won't necessarily see at all. The copy numbers of these HMGR
encoding genes seemed to vary between plants creating some variation to the
results. But the feeling that I got from those experiments is that with potato
at least you can't say the exact number of those isogenes just based on PCR-
reactions. Just by playing around with different conditions and creating a
set of slightly different primers you might be able to get more reliable
results. Hopefully it works!
Good luck!
Marko
Marko Laine (((((
Univ. of Turku, Dept. of Botany (-O-O-)
Biocity 6A -----oOOo--(_)--oOOo------
20520 Turku
Finland
Malaine at polaris.utu.fiMalaine at sara.cc.utu.fi